Pituitary hormone prolactin (PRL), which promotes growth and survival of mammary epithelial cells and plays a key role in breast tumorigenesis, acts through PRL receptor (PRLr). PRL signaling is controlled via down regulation of PRLr in response to the ligand via the mechanisms that are yet largely unknown. Our preliminary data suggest that (i) the bTrCP E3 ubiquitin ligase limits the extent of PRL signaling via PRLr ubiquitination and lysosomal degradation that depends on PRLr phosphorylation on Ser349, (ii) glycogen synthase kinase 3 (GSK3) is implicated in PRLr phosphorylation on Ser349, and (iii) this Ser349 phosphorylation as well as PRLr-bTrCP interaction and the proteolysis of PRLr are impaired in breast cancer cells. We hypothesize that GSK3-dependent phosphorylation of PRLr on Ser349 leads to the recruitment of bTrCP followed by ubiquitination, endocytosis/sorting and lysosomal degradation of PRLr to limit the extent of cellular responses to PRL. In breast cancers, oncogenic signaling-induced aberrations in PRLr phosphorylation and turnover might augment the extent of PRL signaling and promote PRL-mediated cell growth and survival. To test these hypotheses, we propose to: (1) Determine how PRLr stability is regulated in breast cancer cells and tissues that may exhibit impaired PRLr phosphorylation on Ser349;(2) Define the motifs required for PRLr endocytosis (internalization and post-internalization trafficking) and the role of bTrcp-dependent ubiquitination in these processes;(3) Determine whether GSK3, via its ability to phosphorylate PRLr on Ser349, plays a role in ubiquitination and degradation of PRLr and in the regulation of the extent of PRL signaling;(4) Determine whether stabilization of PRLr results in augmentation of the extent of responses to PRL in breast cells in vitro and in mice. Significance: these studies are important for our understanding of the events that occur in breast cancers and, therefore, are relevant to the mission of NCI. Completion of these experiments will potentially: (i). identify PRLr stabilization as a novel event in breast tumorigenesis;(ii) gain insight in the mechanisms underlying down regulation of hormone receptors and expand our knowledge on the mechanisms regulating the cellular responses to PRL, (iii) shed light on the role of PRLr stability in pathogenesis of breast cancer and offer new means for altering the levels of PRL-dependent survival of breast cancer cells.